This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Why do defects in molecular machines (AAA+ ATPases) cause disease? These protein machines convert ATP hydrolysis into mechanical work. Both human cells and disease causing pathogens use this work to physically manipulate proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. We do not know how these molecular machines convert ATP hydrolysis into mechanical work. Our research focuses on one subset of AAA+ ATPAses, the bacterial-enhancer-binding proteins (EBPs) which use their ATPase activities to regulate transcription of genes needed for harmful activites (diseases, crop damage) or helpful ones (nitrogen fixation, environmental remediation, hydrogen or other metabolite production). In prior projects, we established two mechanisms for regulating the EBP ATPases, defined structural changes occuring in their catalytic cycle, and are addressing the underlying mechanism via structure function studies of mutant forms of ATPase. A fascinating yet unexpected outcome was observed upon titrating ADP-BeFx and recording changes in the SAXS profile of the NtrC1 ATPase. Two distinct phases of conformational change were observed. The first involved reducing Rg from 47 [unreadable] to 42 [unreadable] as about half of the available nucleotide binding sites became occupied. The second phase, only occurring after the first transition was completed and additional nucleotide binding sites became occupied, involved change in the SAXS profile at higher Q values.